One step removal of unwanted molecules from circulating blood

ABSTRACT

The present invention provides a method, and resulting product, for the removal of unwanted molecules from a host&#39;s blood using a one-step procedure. The unwanted molecules may be anti-A blood protein and/or anti-B blood protein antibodies that would otherwise cause host rejection of transplanted organs or tissues from a source having a different ABO blood type. The unwanted molecules may also be excess antibodies, or virions, present in a diseased host.

[0001] This application is a Continuation of Ser. No. 09/566,510, filedMay 8, 2000, which is incorporated herein by reference.

FIELD OF INVENTION

[0002] This invention relates to technology for reducing the presence ofunwanted molecules, including those related to disease states and thoseinvolved in rejection of transplanted organs and tissue, a host's blood.In particular, the invention discloses a method and a system forreducing the presence of unwanted molecules, such as anti-A and anti-Bantibodies, by a one step removal process. It also discloses bloodsubstantially of unwanted antibodies, antigens, and the like.

BACKGROUND OF THE INVENTION

[0003] Traditionally, organ or tissue transplantation requires ABO bloodtype compatibility in order to prevent graft rejection. Normally, thehost's blood contains circulating antibodies against foreign blood typeantigens. Transplantation across these ABO blood groups leads tohyperacute rejection of the graft within the first 24 hours (Kuby J:Immunology. New York, W. H. Freeman and Company, 1997). Circulatingantibodies bind to blood antigens present in red blood cells, epithelialcells and endothelial cells found in the graft organ or tissue. Theseantibody-antigen complexes activate the complement system of the host,resulting in infiltration of neutrophils into the graft organ or tissue.The neutrophils release lytic enzymes that destroy the graft endothelialcells, providing a surface of injured tissue to which platelets canadhere. Massive blood clots form within the capillaries, and this wholeinflammatory reaction prevents vascularization.

[0004] Current treatments to reduce rejection include administering aregimen of immunosuppressant drugs before and after the transplantationsurgery. Studies have been performed on methods that remove antibodiesspecific to ABO antigens. These methods have also shown beneficialeffects in reducing hyperacute rejection of the transplanted organ ortissue. These methods are important because they may lead to a methodwhich will relax the requirement of donor/recipient ABO compatibility,which in turn can greatly expand both the living donor and cadaver organor tissue pools.

[0005] Current techniques to remove the ABO antibodies include plasmaexchange combined with intravenous administration of soluble ABOantigens (Alexandre G P J, et al., Neth J Med, 28:231-234, 1985);separating plasma from the whole blood by either centrifugation ordouble filtration plasmapheresis (DFPP) followed by immunoadsorptionusing concentrated red blood cells (Slapak M, et al., Transplantation31:4-7, 1981); and DFPP followed by column immunoadsorption of anti-Aand B antibody using A and B antigen bound to silica beads (Tanabe K, etal., Transplantation Proceedings, 27(1) 1020-1023, 1995).

[0006] These prior art methods have serious problems which haveprevented their adoption as the standard of care. First, there is therisk of infection. Because plasma exchange by centrifugation requiresreplacement by plasma protein solution, risk of viral transmission ispresent. Moreover, these techniques described above involve firstseparation of plasma from whole blood then an additional procedure toremove ABO antibodies from the plasma. Separated plasma can then bestripped of pre-existing anti-A and B antibodies by immunoadsorptionwith ABO antigens linked to silica beads on a column.

[0007] A study on renal transplantation has shown that ABO-incompatiblegrafted patients who received one or two sessions of DFPP and three orfour sessions of column immunoadsorption showed no significantdifference in survival rates when compared to patients who received anABO compatible graft (Tanabe, supra). Additionally, one case has beenreported in which hyperacute rejection following accidentalABO-incompatible renal transplant was reversed using plasmapheresisfollowed by immunoadsorption with red blood cells (Slapak, supra).

SUMMARY OF THE INVENTION

[0008] This invention provides a method and system for reduction of ahost's rejection of a non-autologous organ or tissue transplant causedby the presence of foreign antigens in and on the organ or tissue. Thisis accomplished by providing a method for one-step removal of antibodiesin the host's blood that are directed to the foreign antigens. Forexample, cross-ABO rejection can be eliminated by removing anti-A and/oranti-B antibodies, in one step, from the host's blood. This is done bymoving blood extracted from the host along a pathway, which isoptionally semi-permeable, having antigen specific to the antibodies,such as antigens that bind to anti-A and anti-B antibodies, attached tothe pathway, and returning the blood to the host internal circulation.

[0009] In another embodiment, this invention provides a method forremoving, in one step, excess antibodies, such as are present in certaindisease states, from a host's blood by moving the blood extracted fromthe host on a pathway, optionally semi-permeable, having antigens oranti-antibodies specific to the unwanted antibodies immobilized in thepathway, and returning the blood to the host's internal circulation.

[0010] In yet another embodiment, unwanted antigen is removed from ahost's blood in one step by moving the blood extracted from the host ona pathway, optionally semi-permeable, having antibodies specific to theantigen immobilized in the pathway, and returning the blood to thehost's internal circulation.

[0011] In another embodiment, this invention provides blood that issubstantially free of undesired molecules, such as anti-A and anti-Bantibodies, wherein A and B are blood type antigens. The undesiredmolecules may also be antibodies associated with a disease statecomprising an excess of antibodies in the blood, virions, and otherundesired antigens.

[0012] In the preferred practice of the invention, a hollow fiber hasattached A and B blood type antigens that are capable of sequesteringthe antibodies specific to A and B antigens from the flowing blood. Inanother preferred practice of the invention, the hollow fiber withattached antigen has semi-permeable pores that allow dialysis orplasmapheresis of the blood to occur at the same time. In a furtherpreferred practice of the invention, the hollow fiber is coupled to aplurality of perpendicular membranes having attached antigen.Alternatively, this plurality of membranes can also be longitudinallyplaced inside and along the length of the fiber. In the most preferredembodiment, the antigens are attached to the wall of the hollow fiber.In further practice of the invention, the hollow fiber can be replacedby a flat membrane in a closed container that the blood can flow alongor pass through. In this embodiment, an optional semipermeable membraneis present to divide the flowing blood from a slurry that will inducethe blood components, such as antibodies, to exchange across themembrane.

[0013] The invention also provides a method to increase the organ ortissue pools available for transplant by removing in one step, from thehost's blood, antibodies specific to foreign antigens present in thetransplanted organ or tissue

[0014] The invention also provides a one-step system for removingantibodies to specific antigens from blood in one step.

[0015] The invention also provides a one-step system for harvestingantibodies to specific antigens from blood.

[0016] In another embodiment, the present invention provides circulatingblood that is substantially free of unwanted molecules, wherein thesemolecules are capable of binding, either specifically ornon-specifically, to a binding partner capable of being immobilized on apathway. In particular, this invention provides circulating blood thatis substantially free of anti-A blood protein and anti-B blood proteinantibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 is a longitudinal cross section view of an antibody removalsystem in accordance with a first embodiment of the present invention.

[0018]FIG. 2 is a longitudinal perspective section view of an antibodyremoval system in accordance with a second embodiment of the presentinvention.

[0019]FIG. 3 is a longitudinal perspective section view of an antibodyremoval system in accordance with a third embodiment of the presentinvention.

[0020]FIG. 4 is longitudinal perspective section view of an antibodyremoval system in accordance with a fourth embodiment of the presentinvention.

[0021]FIG. 5 is a top perspective view of an antibody removal system inaccordance with a fifth embodiment of the present invention.

[0022]FIG. 6 shows the results of an assay using the method of thisinvention to remove anti-A and anti-B antibodies from blood.

[0023]FIG. 7 shows the results of an assay using the method of thisinvention to remove anti-A antibodies from blood, showing the highcapacity of the product.

[0024]FIG. 8 shows the results of an assay using the method of thisinvention to remove anti-B antibodies from blood showing the highcapacity of the product,

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] This invention provides a method and a system for one stepremoval, from the host's blood, of antibody specific to foreign antigenspresent in a transplanted organ or tissue. This is done by moving bloodextracted from the host along an enclosed pathway such as a hollow fiberor flat dialyzer comprising bound or immobilized specific antigen, andreturning the blood to the host internal circulation. The bloodcomponents are dialyzed across the membrane of the pathway, while at thesame time antibodies are removed from the blood through binding to theimmobilized antigen. The binding can be specific, as when the antigensare chosen to be the specific binding partners of the antibodies, ornonspecific, as when a general binding molecule such as protein A orprotein G is used to bind the antibodies.

[0026] The antibodies, along with undesired small molecules (urea,creatinine, ammonia), are thus removed from the host's blood.Additionally, these antibodies can be collected by releasing them fromtheir binding partners.

[0027] Expanding on this technique, the invention also provides a meansfor removing other unwanted molecules from a host's blood. For example,virions present in the blood due to a viral infection of the host can beremoved by utilizing immobilized antibodies, either monoclonal orpolyclonal, to the virion.

MATERIALS

[0028] Enclosed Pathway

[0029] The present invention comprises an enclosed pathway that allowsthe flow of blood and the trapping of one of the binding partners of abinding pair, such as an antibody and an antigen. The device can be madeout of a variety of substances, including but not limited tonitrocellulose, cellulose, nylon, plastic, rubber, polyacrylamide,agarose, poly(vinylalcohol-co-ethylene), and the like, and combinationsthereof. The material is preferably semi-permeable to allow the passageof small molecules out of the pathway.

[0030] The device can be formed in a variety of shapes, including butnot limited to a flat dialyzer, a semi-permeable membrane, asemi-permeable hollow fiber, a coil, a dialysis membrane, aplasmapheresis filter, and multiples and combinations thereof.

[0031] The preferred embodiment as shown in FIG. 1 uses a semi-permeablehollow fiber 1 for commercial dialysis with the antigen 3 attached tothe wall 4 of the tubing with or without a linker molecule, for examplePEG (polyethylene glycol), connecting one to the other. Use of dialysismembranes with attached antigen allows direct membrane immunoadsorptionof the specific antibody 5 and plasmapheresis to occur at the same time.

[0032] Alternatively, other anchors for the immobilized binding partnercan be used alone or in combination. For example, the hollow fiber 1 canhave a plurality of flat membranes 9 that are placed longitudinallyalong the fiber length (FIG. 3) or perpendicular to the fiber (FIG. 2).The antigens 3 which are non-diffusively linked to this plurality ofmembranes 9, sequester the specific antibodies 5 from the blood as theypass along the hollow fiber 1. The membranes 9, preferably hi-fluxmembranes, allow blood cells and components to pass through so that noclogging occurs. The tubes themselves may be dimpled, twisted, orotherwise modified to increase mixing and binding of pathogen andantigen.

[0033]FIG. 4 shows another embodiment of the hollow fiber 1 where theantigens are linked to free floating permeable spheres 11 locatedinbetween the plurality of membranes 9. These spheres are trappedbetween the hi-flux membranes because of their size. The antigens 3 onthe spheres 11 sequester the specific antibodies 5, thus removing themfrom the blood. Air or other non-toxic gas may be added at a lowerelevation as small bubbles to further mixing and binding, and then thegas can be removed with a standard bubble trap at a higher elevation(not shown). The gas-induced mixing can occur on either the shell sideor the tube (lumen) side.

[0034]FIG. 5 shows another embodiment of the invention, where theantigens 3 are attached to flat semi-permeable membranes 13 of a flatplate dialyzer 15 instead of a hollow fiber. Blood plasma (as shown bydownward arrows 17) passes through the membrane by convection but thespecific antibodies are retained at the membrane. The blood travelsalong the pathway, continuously or temporarily interrupted, from left toright in the figure.

[0035] Binding Pair

[0036] This invention can be used with any binding pair, including butnot limited to an antigen and an antibody, a receptor and ligand, ananti-antibody and an antibody, or binding portions of these molecules.By the term “binding portions” is meant any portion of the molecule thatis capable of binding, either specifically or non-specifically, to apartner molecule so as either to be removed or to remove the bindingpartner from the blood.

[0037] In the preferred embodiment of the invention, the ABO blood groupantigens are bound to the lumenal surface to remove their correspondingantibodies from blood. The antigen/antibody binding pair can be reversedwherein the antibody is bound to the luminal surface and the antigen isremoved from the blood. Other antibodies, anti-antibodies, and antigens,such as major histocompatibility complex (MHC) molecules, or parts ofthese molecules, can be used to trap antibodies specific to thesemolecules. The antigen/antibody pair can further be replaced with anymembers of sets of binding pairs that would have specific affinities.Examples are ligands and receptors with some specificity to a pathogen.

[0038] Substance A and B antigens can be procured from DadeInternational in Switzerland (trade name: Neutr-AB). This mixture ofSubstance A and B antigen can be from a variety of natural sources,including but not limited to cows, pigs, horses and humans. Theseantigens, in their most reduced form trisaccharides, can also be madesynthetically. A higher affinity for the antigen will exist when theantigen matches the original antigen to which the antibodies wereproduced. Likewise, the more purified the antigen is, the stronger thereaction.

[0039] The more antigen is present, immobilized directly on the lumenalsurface or attached by a linking molecule in the enclosed pathway, themore specific antibody can be removed from the flowing blood. Likewise,the larger the surface area of the coated membrane, the higher thecapacity for binding the desired antibody. For instance, 100 mg ofantigen non-diffusively linked to a hollow fiber can significantlyreduce the anti-A and anti-B titers of 300 to 400 ml of blood with fromaverage to high titer. FIG. 6 shows the capacity of a modified hollowfiber to sequentially process 100 ml of banked human blood. Titer isdetermined by using a standard hemagglutination assay. This shows thatmembrane-bound antigen can specifically remove anti-A and anti-Bantibodies, and that this removal takes place in the first 15 minutes offlow (about 3 passages of the blood over the membrane), regardless oforiginal titer. Alternatively, one antigen type, such as Substance A orB, can be used.

[0040]FIG. 7 shows the capacity of filters modified with A antigen foranti-A antibodies. FIG. 8 shows the same using B antigen for anti-Bantibodies. Consecutive samples of blood were passed over the membraneuntil the membrane was saturated. At this point the titer of antibody inthe blood samples no longer decreased upon passage over the membrane.The anti-A coated membrane had a capacity of around 300-400 ml. ofaverage to high titer blood. The anti-B coated membrane had a capacityof around 600 ml.

[0041] Further purification of the standard antigens leads to at least asix fold increase in capacity to remove anti-A and anti-B antibodies permg of antigen. Purification is achieved by removing components havingmolecular weight below 12,000 daltons from the commercially availableantigen solution by dialysis. For example, the anti-A antibody capacityof a dialysis filter modified with approximately 40 mg of purifiedantigen reduced the anti-A titer of each of six 150 ml blood samples to2 or below. The standard non-purified antigen-modified filter reducedthe anti-A titer of the first sample from 32 to 8, and caused no titerreduction of the other five samples. The results were similar for theanti-B antibodies. Hence we expect that a dialysis filter modified with100 mg of purified may be able to significantly reduce the anti-A andanti-B titers of 1.8 to 2.4 L of average to high titer blood.

[0042] Linking; of Binding. to the Enclosed Pathway

[0043] The antigen, antibody, binding pair member, ligand, or bindingparts thereof, can be linked to the enclosed pathway by a variety ofstandard linking techniques, including but not limited to chemicalmodifications, covalent bonding, strong ionic or hydrogen bonding, useof a linker, etc. The preferred method uses standard cyanogen bromide(CNBr) linking which starts by treating the enclosed pathway with CNBrfollowed by incubation of the antigen and the modified pathway. TheN-terminus of the antigen protein will covalently attach to the CNBrlinker. Other compounds for treating the enclosed pathway include, butare not limited to, hydrogen peroxide, sodium periodate,epichlorohydrin, 1,4-butanedioldiglycidol ether, cyanuric chloride,carbonylkdiimidazole, substituted sulfonyl chloride, or fluoromethylpyridinium salts, and antigen applied in the same way. Standard chemicallinkers such as avidin and biotin can also be used.

PROCESS

[0044] Filtration

[0045] Filtration of unwanted molecules from blood can be achieved usingstandard kidney dialysis type equipment which removes blood from one armand returns it to the other. Alternatively, any pumping system connectedto the patient at two sites, so as to draw blood from one site andreturn it to the other, will work. The blood is passed through theenclosed pathway having immobilized binding partners. The bindingpartners sequester the unwanted molecules as they move along. Severalpasses of the blood along the pathway might be required to completelyremove the specific unwanted molecules.

[0046] The flow rate of blood moving through the pathway must be fastenough to prevent coagulation, yet not so fast as to damage the bloodcells. Examples of ranges are from about 10 to about 1000 ml of bloodper min., preferably between about 50 and about 750 ml/min. and mostpreferably the flow rate for removal of antibodies using the inventionis between about 100 and about 500 ml/min. Heparin can also be added tothe blood to prevent coagulation. Processing of an entire host's bloodvolume (˜5L) would require approximately 2.5 hours to achieve completeremoval of antibodies or other undesired molecules from the blood.

[0047] The flow can be continuous. Alternatively, the flow can beinterrupted to increase the interaction of the unwanted molecules withtheir immobilized binding partners. Likewise, the shape of the devicehaving the immobilized binding partners can be such that it willencourage some swirling and/or backflow to increase the interaction timebetween the unwanted molecules and the immobilized binding partners.

USES

[0048] The current invention can be used in reducing organ or tissuetransplant rejection by removing specific antibodies against foreignantigens found in the transplanted organ or tissue and providingcirculating blood substantially of these antibodies. The invention canalso be used as part of a quantitative assay for specific antibodiesfound in the blood. For example, a whole body assay for the titer ofanti-A and anti-B antibodies can be performed. First, the antibodiesfrom the blood can be removed by the filtration described above. Second,the bound antibodies are released by competing with free floatingantigens or with other very low ionic strength buffers to preventbinding. Third, released antibodies can be titered using a method suchas a hemagglutination assay.

[0049] Also, the invention can be used to preparatively purify specificantibodies from the blood without the need to plasmapherese. The stepsare similar to the quantification assay described above.

[0050] Additionally, the invention can be used to remove excess amountsof antibodies present in the blood.

[0051] Further, the invention can be used to identify, quantify and/orremove other molecules having binding partners, such as virions orligands, from the host's blood.

What is claimed is:
 1. A method for reducing the presence of unwantedmolecules in a host's blood in a single step, comprising: a) extractingblood from the host; b) moving the extracted blood in a continuous ortemporarily interrupted flow along an enclosed pathway, wherein thepathway has immobilized binding molecules specific to the unwantedmolecules attached along the pathway, and the binding molecules arecapable of binding to the unwanted molecules in the blood of the host;and c) returning the blood to the host's internal circulation.
 2. Amethod in accordance with claim 1 wherein the unwanted molecules areantibodies specific to foreign donor antigens present in a transplantedorgan or tissue.
 3. A method in accordance with claim 1 wherein theunwanted molecules are virions or sub-particles thereof.
 4. A method inaccordance with claim 1 wherein the unwanted molecules are antibodiesassociated with a disease state comprising an excess of antibodiespresent in the blood.
 5. A method in accordance with claim 1 wherein thepathway is selected from the group consisting of a flat dialyzer, asemi-permeable membrane, a semi-permeable hollow fiber, a coil, dialysismembrane, a plasmapheresis filter, and combinations thereof.
 6. A methodin accordance with claim 5 wherein the pathway is at least partiallydimpled, twisted, or otherwise modified to increase mixing and bindingof unwanted molecules.
 7. A method in accordance with claim 5 whereinthe pathway is composed of a material selected from the group consistingof nitrocellulose, cellulose, nylon, plastic, rubber, polyacrylamide,agarose, poly(vinylalcohol-co-ethylene), and combinations thereof.
 8. Amethod in accordance with claim 1 wherein the specific binding moleculesare selected from antigens, antibodies, anti-antibodies, ligands,receptors, binding portions thereof, and combinations thereof.
 9. Amethod in accordance with claim 8 wherein the antigens are selected fromthe group consisting of A blood type antigens, B blood type antigens,protein A molecules, protein G molecules, major histocompatibilitycomplex molecules, binding portions thereof, and combinations thereof.10. A method in accordance with claim 1 wherein the antigens areattached along the semi-permeable pathway by a process selected thegroup consisting of chemical modifications, covalent bonding, strongionic bonding, hydrogen bonding, and use of a linker.
 11. A method inaccordance with claim 10 wherein the chemical modification isaccomplished by treatment with a compound selected from the groupconsisting of cyanogen bromide, hydrogen peroxide, sodium periodate,epichlorohydrin, 1,4-butanedioldiglycidol ether, cyanuric chloride,carbonyldiimidazole, substituted sulfonyl chloride, and fluoromethylpyridinium salts.
 12. A method in accordance with claim 1 wherein theantigens are attached along the enclosed pathway by avidin or biotinlinkers.
 13. A method in accordance with claim 5 wherein the specificantigens are attached to the wall of a semi-permeable hollow fiber. 14.A method in accordance with claim 13 wherein the hollow fiber is coupledwith a plurality of enclosed parallel membranes.
 15. A method inaccordance with claim 14 wherein the plurality of enclosed parallelmembranes are arranged perpendicular to the hollow fiber.
 16. A methodin accordance with claim 14 wherein the plurality of enclosed parallelmembranes are longitudinally arranged inside and along the length of thehollow fiber.
 17. A method in accordance with claim 14 wherein theplurality of parallel membranes have antigens attached to them.
 18. Amethod in accordance with claim 1 wherein the blood is moved into, alongan enclosed pathway within, and out of a container, wherein thecontainer is closed except for entrance and exit openings, and whereinthe container comprises a) a slurry having immobilized binding partnersattached to the slurry particles, and b) at least one flatsemi-permeable membrane, the membrane dividing the flowing blood fromthe slurry, wherein the blood components can exchange with the slurryacross the membrane.
 19. A method in accordance with claim 18 whereinthe flat semi-permeable membrane is made of a material selected thegroup consisting of nitrocellulose, cellulose, nylon, plastic,polyacrylamide, agarose, poly(vinylalcohol-co-ethylene), and rubber. 20.A method in accordance with claim 18 wherein at least one additionalmembrane is present in the portion of the container where the bloodflows, and wherein the additional membrane or membranes are arrayedeither perpendicularly or horizontally to the blood flow. 21.Circulating blood substantially of unwanted molecules, wherein themolecules are capable of specific or non-specific binding to a bindingpartner.
 22. The circulating blood in accordance with claim 21 whereinthe unwanted molecules are anti-A blood protein and anti-B blood proteinantibodies.